Cell membranes are self assembling boundaries of a living cell or of internal compartments thereof, defined by a bilayer architecture based on lipids. Membrane proteins are large, amphiphilic moieties with subtle function-structure dependencies. Membrane proteins can be associated with the membrane to different degrees. A classical differentiation classified membrane proteins according to the conditions required to solubilise the same. “Peripheral” membrane proteins can be dissociated from the membrane by relatively mild conditions such as increased ionic strength or a change in pH. “Integral” membrane proteins can only be dissociated from the membrane by means of detergents or organic solvents. Such “integral” membrane proteins include proteins that are now known to span the entire membrane, proteins known to be partially embedded within an outer portion of the membrane such as prostaglandin H2 synthase-1, but also some proteins that interact with the membrane via hydrophobic posttranslational modifications such as heterotrimeric G proteins.
It is an extremely challenging task to provide a biomimetic structure that allows preserving the functional structure of a membrane protein, which has a portion that is included in a cellular membrane in vivo. The conventional detergent-based isolation methods of membrane proteins and addition to surfaces results in their random orientation after incorporation. The conventional approach does not allow a complex membrane protein to be incorporated in a form which maintains the availability and functional presentation of the protein. This is critical for any application, in particular, biosensing and drug discovery. Furthermore, it is difficult to maintain the robustness of a conventional membrane layer on a surface when it is formed from traditional phospholipid materials.
Membrane proteins play a crucial role in many functions involving interaction, including communication, with a cell's ambience, which are usually important to an individual cell as well as to an organ and an organism that includes the same. Prominent examples are cellular signalling, selective transport of components into and out of the cell or cell-cell interactions. About 50% of the drug targets are membrane proteins. Membrane proteins are for example involved in visual perception, olfaction and taste reception. Important examples of proteins with membrane spanning domains, G protein-coupled receptors (GPCRs) tyrosine kinase receptors, receptor channels (usually termed ionotropic receptors, e.g. glutamate receptor channels) and TOL-like-receptors are key signaling receptors in cells. Despite their importance, detailed functional studies of membrane proteins are still scarce because of their sensitivity and notorious difficult procedures of isolation and functional re-constitution.
While a variety of assay techniques is meanwhile available for screening of membrane receptors (for an overview see e.g. Cooper, M A, J. Mol. Recognit. (2004) 17, 286-315), a reconstitution system for easily handling the same is still unavailable. Strategies available in the art to analyse the functions of membrane proteins include membrane-based model systems as well as cell-based model systems. The standard protocols typically followed have remained essentially unchanged over the last decades and involve either vesicles formed from detergents or fragments obtained from biological cell membranes (e.g. ibid.).
In an approach of mimicking a biological membrane situation, vesicles of synthetic amphiphilic linear block co-polymers with polar terminal groups have been formed (cf. e.g. Disher, D E, et al., Science (2002) 297, 967-973). In a further approach, an artificial membrane of a layer of dimyristoylphosphatidylethanolamine and a layer of phosphatidyl-choline has been formed on a gold substrate (international patent application WO 2007/048459, cf. FIG. 1). To allow analysing the function of membrane proteins encompassed by the membrane, direct contact between membrane proteins and the solid support is avoided by means of a hydrophilic peptide layer, acting as a macromolecular spacer (ibid.). Synthesis and incorporation of membrane proteins onto this tethered bilayer membrane architecture have been monitored by Surface Plasmon enhanced Fluorescence Spectroscopy (SPFS), immuno-assays (Western Blot), Surface Enhanced Infrared Spectroscopy (SEIRAS) and radioactive labeling. With this system, direct synthesis of membranes in an artificial environment, as well as characterization of membrane proteins on a molecular level can be achieved. However, mechanical strength is compromised as a result of the fragile nature of the membrane formed by phospholipids. In addition, it is difficult to incorporate this technology into a multi-well format. Therefore there remains a need for an alternative approach of providing an artificial membrane.
Accordingly, it is an object of the present invention to provide a robust architecture of or with an artificial membrane that is well suited for assay development, analysis and screening purposes.